The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that in addition to photosynthesis are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression.
DNA sequence and biochemical data reveal a similarity of the plastid organelle's transcriptional and translational machineries and initiation signals to those found in prokaryotic systems. In fact, plastid derived promoter sequences have been reported to direct expression of reporter genes in prokaryotic cells. In addition, plastid genes are often organized into polycistronic operons as they are in prokaryotes.
Despite the apparent similarities between plastids and prokaryotes, there exist fundamental differences in the methods used to control gene expression in plastids and prokaryotes. As opposed to the transcriptional control mechanisms typically observed in prokaryotes, plastid gene expression is controlled predominantly at the level of translation and mRNA stability by trans-acting nuclear encoded proteins.
Previous studies directed to stable transformation of plant chloroplasts have relied on homologous recombination to incorporate desired gene constructs into leaf plastids. In this manner, transgenic plants homoplastic, or near-homoplastic, for a recombinant DNA construct may be obtained. However, a major drawback to genetic engineering for plastid gene expression is the lack of tissue specific and/or developmental regulation mechanisms to control the timing and/or sites of expression of the desired gene products. Since the entire complement of plastid organelles in the transgenic plants are transformed, the integrated construct is expressed in all plastid containing plant tissues.
A mechanism for controlling expression of sequences inserted into plastids would be useful for optimum modification of plastid pathways which occur in particular tissue types, such as the starch and fatty acid biosynthesis pathways in potato tubers or oilseeds, respectively, flower color pathways, fruit ripening related reactions in various fruit plastids, and pathways which can be targeted to produce herbicide resistance in green plant tissues. In addition, unregulated modification of existing plastid metabolism, for example by reducing expression of a native plastid gene using antisense constructs, and/or introduction of new biochemical pathways could result in the inability to obtain viable plants. For example, alteration of certain pathways in vegetative tissues at an early developmental stage, such as would be observed using non-regulated E. coli or chloroplast gene promoters, could result in the production of detrimental end products and thus limit the ability to obtain transgenic plants. However, if the gene for a desired biochemical reaction could be programmed for expression only at a particular stage of development, the reaction could be controlled so as to produce the desired product at the desired period, for example when there is sufficient plant biomass to harvest. In this manner, substantial quantities of the desired end product may be obtained.